A number of diseases are associated with alterations in cell death pathways. Excessive cell death leads to progressive organ dysfunction, such as ischemic diseases and neurodegeneration. Inadequate cell death is a hallmark of cancer, and contributes to certain autoimmune diseases. Furthermore, the beneficial or detrimental effects of many drugs can be attributed to their effects on cell death. Therefore, non-invasive methods for assessing cell death would provide clinicians with critical information on disease activity and therapeutic efficacy. To date, there are no probes approved for clinical imaging of apoptosis. We have unexpectedly found that a variety of dehydrogenase proteins specifically accumulate in cells undergoing apoptosis and necrosis. For example, recombinant lactate dehydrogenase (LDH), when fluorescently labeled, specifically stains both early apoptotic and late apoptotic/necrotic cells. While LDH is known to be an intracellular protein that leaks out of dying cells, we find that exogenously applied LDH- based probes specifically accumulate within dead cells. We have found that LDH probes have several advantages over Annexin V, which is currently in clinical trials for imaging of apoptosis, including no requirement for calcium and no effect of serum proteins on staining. These characteristics suggest that LDH-based probes may be superior to Annexin V for in vivo imaging of cell death. In this proposal, we seek to establish an optimized LDH-based probe for imaging apoptosis. We will first establish the in vitro and in vivo characteristics of a full length LDH radioligand for binding to apoptotic cells and in vivo imaging. In the first Specific Aim, we will then use systematic mutagenesis to define the structural elements necessary and sufficient for LDH-based probes to stain apoptotic cells. In the second Specific Aim, we will develop a series of LDH-based radioligand probes, and characterize their biodistribution in mice. In the third Specific Aim, we will use three mouse models of apoptosis to identify an optimized LDH-based probe for PET-imaging of cell death. Finally, the performance of an optimized LDH probe will be directly compared to that of Annexin V for iin vivoi imaging of cell death. Together, success in these studies would provide proof-of-concept for imaging apoptosis using novel LDH-based probes, and would establish the rationale for proceeding with human clinical testing. PUBLIC HEALTH RELEVANCE: The ability to non-invasively image cell death would provide a critical tool for diagnosing multiple diseases and monitoring the efficacy of drug treatments. No probes have yet been approved for the clinical imaging of apoptosis, underscoring the great unmet need for new sensitive and specific probes. The proposed studies seek to develop an entirely novel method for imaging apoptosis, and to bring this probe to readiness for human clinical testing.